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目的筛选ataxin3的相互作用蛋白并进行互作结构域分析,探讨ataxin3的功能和脊髓小脑型共济失调Ⅲ型/马查多约瑟夫病的发病机理。方法应用酵母双杂交系统3,从成人脑cDNA文库中筛选和鉴定突变型ataxin3的互作蛋白,构建ataxin3蛋白羧基端Bait质粒,进行互作结构域分析。应用激光共聚焦显微镜观察ataxin3与所筛到的互作蛋白在哺乳动物细胞中的共定位情况。结果分离获得5个新的ataxin3互作蛋白,视紫红质二磷酸鸟苷解离抑制因子α、苏素1、氨氯吡嗪脒敏感性神经元阳离子通道2和2个未知新序列。结构域分析显示除1个未知蛋白与ataxin3蛋白羧基端互作外,其余4个均与氨基端互作。在SHSY5Y细胞的细胞核内,野生型ataxin3与苏素1共定位,突变型ataxin3所形成的核内蛋白聚合体也与苏素1共定位。结论发现1个未知蛋白可能与ataxin3蛋白羧基端互作,苏素1可能与ataxin3蛋白氨基端互作,苏素化可能参与了ataxin3蛋白的翻译后修饰和脊髓小脑型共济失调Ⅲ型/马查多约瑟夫病的发病过程。
Objective To screen the interacting proteins of ataxin3 and analyze the interaction domains of ataxin3 to explore the pathogenesis of ataxin3 and Spinocerebellar ataxia type Ⅲ / Machado Joseph disease. Methods The yeast two-hybrid system 3 was used to screen and identify the mutant ataxin3 interacting proteins from adult brain cDNA library to construct the carboxy-terminal Bait plasmid of ataxin3 protein for analysis of the interaction domain. Confocal laser scanning microscopy was used to observe the localization of ataxin3 and the screened interacting proteins in mammalian cells. Results Five novel ataxin3 interacting proteins, guanosine diphosphate guanosine diphosphate decarboxylase inhibitor α, suprostin 1, amiloride - sensitive neuronal cation channel 2 and two unknown new sequences were isolated. Domain analysis showed that all but one unknown protein interacted with the carboxy terminal of the ataxin3 protein, and the other four all interacted with the amino terminal. In the nucleus of SHSY5Y cells, the wild-type ataxin3 co-localized with Su Su 1, and the nuclear protein complex formed by the mutant ataxin 3 also co-localized with Su Su 1. CONCLUSIONS: One unknown protein may interact with the carboxyl end of ataxin3 protein, Su-1 may interact with the amino end of ataxin3 protein, and threonine may be involved in the posttranslational modification of ataxin3 protein and Spinocerebellar ataxia type Ⅲ / Joseph disease process.